- Title
- In vivo investigations into the effects of pathological conditions including diabetes on lymphatic function and wound healing
- Creator
- Abdulla, Yusupjan
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2016
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Diabetes is a fast growing chronic disease all over the world including Australia. The 1999–2000 Australian Diabetic study indicated that 880,000 Australians had diabetes with most exhibiting type 2 diabetes (96%), and indicated this number increased by 0.7 %/year in a 2011-2012 follow-up study. Foot ulcer(s), generally referred to as diabetic foot, is one of the most severe diabetic complications, which combined with infection may lead to foot amputation. Lymphatic function was investigated in several pathological conditions (i.e. type 1 diabetic mellitus, inflammation and L-type Ca2+ channel blocker-treated oedema). Wound healing was also studied, as it has a close link to diabetes and possibly to lymphatic function. Short- and long-term hyperglycaemia in diabetes has multiple effects. The consequences of hyperglycaemia on the lymphatic system, including the propulsion of lymph, has not been well-studied. We established hyperglycaemia in rats by injecting streptozotocin (STZ), which caused rats to rapidly become hyperglycaemic (from 7 to 18 mmol/l in 1 day) and lose weight (9% reduction 3 days later). We tested lymphatic function 3-5 days after STZ treatment by near infrared (NIR) lymphatic imaging using the dye indocyanine green (ICG) injected into the hind foot of urethane-anaesthetised rats. Lymphatic function measured by contraction frequency and ICG arrival time in exposed groin lymphatic vessels did not differ in control and hyperglycaemic animals. The effect of hyperglycaemia in the initial stage of diabetes on contraction frequency and first arrival time is subtle or compensated such that lymphatic vessels function normally. Understanding and treating diabetic wounds remains a major challenge. Numerous studies demonstrate that hyperglycaemia hinders wound healing through multiple pathways. It is also known that glibenclamide, an anti-diabetic drug, slows down the healing process in various non-diabetic experimental wound models. The direct effect of hyperglycaemia and glibenclamide on wound healing is poorly understood. Streptozotocin (STZ) -treated rats, which were markedly hyperglycaemic even 1 day after treatment, had a small circular skin wound made on an upper hind foot under anaesthesia on day 3 after STZ treatment. A corresponding control group had the same skin wound made. Wound healing was compared for 3 conditions (control, STZ treatment; STZ treatment with glibenclamide cream applied to the same hind limb) by taking daily images of the wound with assessment of healing made by measuring parameters such as: the percentage wound area; the average time for the wound to close to a specified percentage; the time to complete wound closure; and the linear advance of the wound edge. We determined that glibenclamide only had a small negative effect on wound healing. This adverse effect is likely to be well compensated by its normal hypoglycaemic role in treatment of type 2 diabetes. Severe systemic infection and resultant inflammation have been shown to suppress rat lymphatic contractile activity and hence the propulsion of lymph through increased cytokines. In clinical medicine, infection and associated inflammation often occur at low to moderate levels. In order to investigate the effects on lymphatic function of a low to moderate level of infection/inflammation, inflammation was induced by injecting a low dose of lipopolysaccharides (LPS at 1.65 mg/kg) into the rat foot. The function of the inguinal-to-axillary lymphatic vessels was assessed by performing near infrared (NIR) imaging after LPS-induction of inflammation, with the level of inflammation assessed by cytokine measurement from blood. Inflammation was induced, as cytokines interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and the chemokine GRO/KC were all significantly increased. In contrast, spontaneous lymphatic contraction frequency and contraction wave velocity were not altered by low dose LPS injection though overall lymph movement, as assessed by the summed distance the waves of contraction moved in a 5 minute period was increased compared to saline control. Increased lymphatic function assessed with 5-minute lymph travel distance (L₅) is possibly consistent with increased oedema caused by the LPS. The KATP channel blocker glibenclamide did not alter inflammation, as the cytokine levels and lymphatic function were not significantly changed indicating KATP channels were not involved in the regulation of cytokine production. Peripheral oedema is one of the common complications of L-type Ca2+ channel blocker drugs in hypertensive patients. We hypothesized that compromised lymphatic function would hinder fluid transportation from the interstitial space due to reduction in lymphatic contractile activity by calcium channel blockade. We tested whether rat lymphatic function is compromised during nifedipine inhibition of L-type Ca2+ channels using near infrared (NIR) imaging of groin vessels. Nifedipine solution was infused 200 µg/kg/min or 400 µg/kg/min intravenously in deeply anesthetised rats in which arterial blood pressure was monitored. Contraction frequency and contraction wave velocity were monitored in vivo using NIR. Although both 200 µg/kg/min and 400 µg/kg/min nifedipine caused significant reduction in arterial blood pressure, lymphatic vessel contraction frequency and contraction wave velocity did not alter during nifedipine infusion. Lymphatic contraction frequency and contraction wave velocity could be obtained in a quantifiable manner through NIR imaging of the rat groin. Nifedipine, an L-type calcium channel blocker, at doses that caused significant reduction in arterial blood pressure, did not reduce lymphatic drainage. Therefore, other factors such as increased filtration in the microcirculation are likely to be a major cause of nifedipine-related peripheral oedema. Finally we note that during the above-mentioned experiments we investigated rat lymphatic contractile activities in different regions (i.e. deep groin, groin and inguinal-to- axillary lymphatics) finding the contractile activity differed in these regions. A new finding made during these studies was demonstration of two lymphatic pathways from the tail injection site to the inguinal lymph node.
- Subject
- rat; lymphatic function; near infrared; inflammation; diabetes; wound healing; LPS; <i>in vivo</i>
- Identifier
- http://hdl.handle.net/1959.13/1314637
- Identifier
- uon:22793
- Rights
- Copyright 2016 Yusupjan Abdulla
- Language
- eng
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| View Details Download | ATTACHMENT02 | Thesis | 21 MB | Adobe Acrobat PDF | View Details Download |